PROJECT SUMMARY This project will investigate mammalian DNA polymerase ?, the defining enzyme for repair of DNA double-strand breaks by polymerase theta-mediated end joining (TMEJ). Despite the biological importance of TMEJ and its relevance to cancer, we know surprisingly little about its molecular mechanisms. Pol ? is a large protein (290 kDa in mammalian cells) with a distinctive arrangement of a helicase-like domain linked to a DNA polymerase domain. In addition to the polymerase domain (PolD), Pol ? possesses a helicase-like domain (HelD) and a connecting central domain (CenD). Structural information is essential for analyzing DNA polymerase mechanisms, especially for a large multi-domain enzyme such as Pol ?. We determined the first crystal structure of the polymerase domain (PolD) of Pol ?, together with DNA and an incoming nucleotide. The structure revealed unique features, which help explain some of the properties of the polymerase. We located 5 insertion loops in the polymerase and pseudo- exonuclease domains. We presented evidence for dimerization of the PolD, which could function during joining of two DNA molecules. This project aims to fill several major gaps in knowledge, needed to help us understand the unique activities and structure of Pol ?: (1) What role do the ?insertion? loops play in mammalian Pol ? (2) What is the structural basis of helicase-like domain (HelD) function? (3) How does end-trimming occur during microhomology selection? (4) How do molecules of Pol ? coordinate repair, using specific interfaces? In addition to gaining a fundamental understanding of the TMEJ mechanism, the research will reveal new targeting opportunities for Pol ? inhibition in cancer therapy. These structural studies will be highly coordinated within the Program Project with the other three Projects with complementary experimental approaches ? single-molecule characterization of molecular function, activity assays using full-length proteins, and cellular studies of repair. Substrates, proteins, and experiments will be designed with Projects 1, 2, and 4 and monitored with feedback via Core A. Protein purification will be supported by Core B, and cell line construction by Core C. Our combined work will provide unparalleled insight into the TMEJ pathway, and how its mechanism impacts its ability to fulfill its biological role. These insights will be critical to our understanding of the contribution of this pathway to genome instability and carcinogenesis, as well as the evaluation of this pathway as a safe and effective target for cancer therapy.